Abstract Chemokine receptor signaling is a critical determinant of primary tumor growth and organ-specific metastases in many common cancers. In particular, our laboratory and others have shown that chemokine receptors CXCR4 and CXCR7 are upregulated on malignant cells in breast and lung cancer. CXCR4 and CXCR7 signaling significantly increases primary tumor burden and enhances survival and proliferation of metastatic cancer cells, all of which adversely affect quality of life and survival for patients. Chemokine receptor signaling in the tumor microenvironment is initiated by stromal cells that secrete chemokine ligands. These ligands bind to cognate receptors on tumor cells, thereby activating downstream effectors that control tumor progression and metastasis. Understanding integrated functions of chemokine receptors CXCR4 and CXCR7 is essential for dissecting molecular regulation of primary and metastatic cancer and effectively targeting these receptors to improve cancer therapy. We hypothesize that molecular imaging reporters can be developed to detect and quantify multiple steps in CXCR4 and CXCR7 signaling in cell-based assays and mouse models of breast cancer. These imaging reporters build upon our expertise in imaging protein-protein interactions in signaling pathways. In aim 1, we will develop a luciferase complementation reporter to analyze extracellular binding of chemokine ligands to CXCR4 and CXCR7 on breast cancer cells. In aim 2, we will construct an imaging reporter system to quantify chemokine receptor complexes on cells and changes in these complexes during signaling. Finally, aim 3 will develop an imaging reporter for intracellular activation of CXCR4 and CXCR7 based on recruitment of the scaffolding protein -arrestin. This research will establish new imaging technologies to investigate integrated functions of CXCR4 and CXCR7 in breast cancer and establish therapeutic targeting of these chemokine receptors in intact cells and living mice. Beyond the current research, each of these reporter technologies is expected to serve as a general method to study the entire family of chemokines and chemokine receptors, extending applications of these imaging techniques to many diseases besides cancer. The significance of this research is that it will develop new techniques to image intercellular and intracellular signaling networks in breast and a wide range of other common cancers. These imaging reporters will advance our knowledge of cell signaling in pre-clinical models of cancer initiation and progression to metastatic disease. Using these imaging technologies also will greatly improve our ability to test and validate new therapeutic agents, which we anticipate will lead to improved treatments for patients with cancer.